JP6143284B2 - Anti-influenza virus agent - Google Patents

Description

  The present invention relates to a compound or salt thereof in which a receptor pseudo-sialog sugar chain moiety having a hemagglutinin binding ability is bound to a hydrophobic moiety and a polymer moiety via a linker moiety, an influenza preventive and / or therapeutic agent containing them as an active ingredient, etc. Involved.

  The World Health Organization (WHO) raised the alert level to the highest phase 6 in response to the global spread of new swine-derived influenza virus (H1N1) and declared a pandemic. Highly pathogenic avian influenza virus (H5N1) has been transmitted from birds to humans in 15 countries around the world, resulting in a fatality rate of approximately 60% and more than 200 deaths. It is.

  Influenza viruses exist in types A, B, and C, and types B and C are mainly isolated from humans, but type A is transmitted between animals and repeats mutations to acquire pathogenicity and adaptability to humans. It is the most widely distributed zoonotic virus in the world. There are two types of spike glycoproteins, the trimeric hemagglutinin (HA) and the tetrameric neuraminidase (NA) in the influenza virus membrane. Influenza viruses are infected by the interaction of their HA with sialoglycans on the host cell surface. The propagated virus cleaves the sialic acid on the cell surface of the host by the sialidase activity of NA and is released from the host cell. There are many subtypes of type A viruses, and H1 to H16 are identified as the types of HA, N1 to 9 are identified as the types of NA, and this combination alone leads to 144 types. Furthermore, these HA types, Even if the NA type is the same, various mutant viruses with different infection routes and symptoms continue to increase (Non-patent Document 1).

  As anti-influenza drugs, neuraminidase inhibitors and M2 ion channel inhibitors are used clinically, but HA function inhibitors have not yet been developed as clinically used drugs. RNA polymerase inhibitors are also under development but have not yet been used clinically. NA inhibitors currently used as anti-influenza drugs are Tamiflu (trade name) (Roche, generic name: oseltamivir phosphate) and Relenza (trade name) (Glaxo Smithkline, generic name: zanamivir hydration) Products), Inavir (trade name) (Daiichi Sankyosha, generic name: laninamivir octanoate hydrate), Rapiacta (trade name) (Shionogi Pharmaceutical, generic name: peramivir hydrate) . NA is an enzyme involved in virus budding and release from infected cells, which is essential for the virus growth cycle. Inhibition of NA prevents infection of other cells. NA is present in both influenza A and influenza B, and its active site homology is high, so NA inhibitors are effective for both types and are considered to be an effective method for treating influenza and have been widely used. . However, there is a problem that an effective therapeutic effect cannot be achieved against mutant strains, and in 2012, Tamiflu resistant strains have already begun to spread worldwide. Therefore, the development of next-generation anti-influenza virus drugs with a completely new mechanism of action is an urgent issue.

  Moreover, influenza HA vaccine is also widely used as a preventive method against influenza. Influenza vaccines can relieve symptoms such as high fever and reduce hospitalization and death due to complications, and vaccination is recommended especially for elderly people and those with underlying diseases. Development of improved vaccines that are more effective is underway, but it is difficult to predict future virus strains every season, and new pandemic pandemics are anticipated. There is a concern that it will not be able to respond to a pandemic because vaccine production is not possible at this stage.

  As an influenza therapeutic agent, Neu5Ac3αF-DSPE, which is a sialic acid derivative, inhibited NA and inhibited H3N2 virus HA, but no inhibitory activity was observed against H1N1 virus HA. In addition, it has been disclosed that the cytopathic effect due to the adsorption of influenza virus to the cell membrane and influenza virus infection was suppressed (Patent Document 1). Further, it is disclosed that a polymer compound composed of a biodegradable polymer containing glycosphingolipid stably binds to and inhibits HA of H1N1 virus (Patent Document 2). Patent Document 3 discloses a novel sialic acid derivative having a sialidase inhibitory activity targeting HA glycoproteins such as human parainfluenza virus. However, since it targets only HA, the effect on influenza mutants is weak. There was a problem of becoming. Therefore, there has been a demand for the development of an effective medicine for preventing and treating influenza without losing the effect on influenza mutants.

  In addition, from the viewpoint of preventing influenza, it is also possible to capture and inactivate influenza virus before entering the body. Currently, it is pointed out that many infectious diseases such as influenza may cause a pandemic in a place where an unspecified large number of people such as airplanes spend a certain amount of time in the same space.

  For this reason, development of the effective material which removes the influenza virus which floats in the air is desired.

JP 2001-131074 A Japanese Patent No. 3390965 JP 2006-241024 A

Antibacterial fungus, vol 39, No. 10, p55-65 (2011)

  An object of the present invention is to provide a drug effective for preventing or treating influenza, a material for removing influenza virus from the environment, and the like without losing the effect on influenza mutants.

  As a result of intensive investigations, the present inventors have found that a compound in which a receptor pseudo-sialoglycosyl moiety having a hemagglutinin-binding ability, a hydrophobic moiety, and a polymer moiety are bonded to the virus surface via a linker moiety. It has been found that it inhibits both HA and NA, and also inhibits the mechanisms such as adsorption of the virus to the cell surface, virus budding, and infection, thereby completing the present invention.

  That is, the present invention provides [1] the following formula (I)

(In the formula, S is a receptor pseudo-sialog sugar chain moiety having hemagglutinin binding ability, L is a linker moiety having a structure of 5 angstroms or more, and C is a carbon that may have a branched structure or a double bond. A hydrophobic moiety which is an alkyl group having a number of 3 to 35, and P represents a polymer moiety having a chain length of 100 angstroms or more) or a salt thereof; [2] a receptor pseudo-sialoglycosaccharide having a hemagglutinin binding ability Wherein the moiety comprises a Neu5Acα2-6Galβ1-4 (3) GlcNAcβ1 structure, or the salt thereof, or [3] the polymer portion is polyglutamic acid, [1] to [2], wherein the compound or salt thereof according to [1] or [2], or [4] the linker moiety is lysine. It relates to compounds or a salt thereof according to any one of].

  The present invention also provides [5] the following formula (II)

Wherein PGA is the formula (III)

Where m and n are integers and (m + 1) n is 50 to 1000. ) Or a salt thereof according to any one of [1] to [4] above, or [6] any one of [1] to [5] above. An inhibitor of influenza virus hemagglutinin function and sialidase, comprising the compound or a salt thereof as an active ingredient, [7] the inhibitor according to [6], wherein the sialidase is neuraminidase, and [8] the above [ Influenza virus infection, adsorption and budding inhibitors comprising the compound or salt thereof according to any one of 1) to [5] as an active ingredient, and [9] any one of [1] to [5] above The present invention relates to a preventive or therapeutic agent for influenza comprising the above compound or a salt thereof as an active ingredient.

  The present invention also provides [10] an influenza virus adsorbent comprising the compound or salt thereof according to any one of [1] to [5] as an active ingredient, and [11] the above [1] to [5]. An adsorbent for influenza virus carrying a compound or salt thereof according to any one of the above, or an influenza virus mutation carrying a compound or salt thereof according to any one of [12] [1] to [5] [13] The present invention relates to a kit for examining the in vivo kinetics of influenza virus containing the compound or salt thereof according to any one of [13] [1] to [5] as an active ingredient.

  According to the present invention, it is possible to provide a compound or a salt thereof that inhibits the hemagglutinin function and sialidase activity of influenza virus and inhibits multiple mechanisms such as virus adsorption onto the cell surface, virus budding, and infection. it can. In addition, since the compound of the present invention or a salt thereof inhibits the function and sialidase activity of the influenza virus, the influenza prevention or treatment is applicable to the emerging resistant strain without losing the effect on the influenza mutant strain. It can be provided as a medicine. Furthermore, by using the compound of the present invention or a salt thereof as an adsorbent or adsorbent for influenza virus, it can be provided as various products having the ability to reduce and remove influenza virus from the living environment.

It is a figure showing an example of the structure of the influenza preventive and therapeutic agent of this invention. It is a figure showing the outline of the example of the synthesis | combining method of the influenza preventive and therapeutic agent of this invention. 1 shows the NMR spectrum of Compound 3. It is a figure which shows the capillary electrophoresis result of sugar chain 1% introduction | transduction polyglutamic acid compound. It is a figure which shows the result of ¹ H-NMR of a polyglutamic acid compound with 1% sugar chain introduced. It is a figure which shows the capillary electrophoresis result of the sugar chain 5% introduction | transduction polyglutamic acid compound. It is a figure which shows the result of ¹ H-NMR of a polyglutamic acid compound with 5% sugar chain introduced. It is a figure which shows the capillary electrophoresis result of the sugar chain 10% introduction | transduction polyglutamic acid compound. It is a figure which shows the result of ¹ H-NMR of a polyglutamic acid compound with 10% sugar chain introduced. The compounds of the present invention are of type A 2009 pandemic strain (A / Narita / 1/2009 (H1N1); top), A Hong Kong type strain (A / Yamaguchi / 20/2006 (H1N1); middle) and A Soviet type ( It is a figure which showed that the sialidase activity was strongly inhibited in all the seasonal viruses of A / Aichi / 75/2008 (H3N2); lower stage). A) The sialidase inhibitory activity of the compound of the present invention at a concentration of 0.07 mg / mL against various viruses, and B) the compound at each concentration step against the seasonal virus A Soviet type (A / Aichi / 75/2008 (H3N2)) Sialidase inhibitory activity and IC ₅₀ are shown. The compound of the present invention (polyglutamic acid compound having a sugar chain introduction rate of 5%) is A) 2009 pandemic strain (A / Narita / 1/2009 (H1N1)), B) seasonal influenza virus strain (A / Yamaguchi / 20 / It is a figure which shows combining with 2006 (H1N1). The compound of the present invention (polyglutamic acid compound with 5% sugar chain introduction rate) is C) seasonal influenza virus strain (A / Aichi / 75/2008 (H3N2)), D) type B clinical isolate (B / Aichi / 3) / 2008). It is a figure which represents typically the influenza virus inhibition mechanism of this invention.

  The compound of the present invention or a salt thereof has the following formula (I)

(In the formula, S is a receptor pseudo-sialog sugar chain moiety having hemagglutinin binding ability, L is a linker moiety having a structure of 5 angstroms or more, and C is a carbon that may have a branched structure or a double bond. It is a hydrophobic moiety that is an alkyl group of several 35 or less, and P represents a polymer moiety having a chain length of 100 angstroms or more) or a salt thereof.

  In the compound of the present invention, the “receptor pseudo sialo-glycan moiety having hemagglutinin-binding ability” may be any as long as it has the ability to bind to influenza virus via hemagglutinin, and specifically, sialosaccharide having hemagglutinin-binding ability. It only has to contain a chain. A sialic sugar chain is a sugar chain compound in which sialic acid (NeuAc), galactose (Gal) and N-acetylglucosamine (GlcNAc) are bound. Sialic acid (NeuAc) is a basic region consisting of galactose (Gal) and N-acetylglucosamine (GlcNAc) (type 1 (Galβ1-3GlcNAc: constituting a type 1 sugar chain) or type 2 (Galβ1-4GlcNAc: type 2) The sugar chain can be linked to α) -6 bond or α2-3 bond. As the receptor pseudo-sialoglycosaccharide moiety of the present invention, sialic acid is bonded to the adjacent glycoside of galactose with an α-glycoside bond, and the binding position between sialic acid and galactose may be the 3rd or 6th position. When administered to humans, it is preferable that sialic acid is bonded to the 6-position of galactose. In order to further enhance the binding property, it is more preferable that a glycoside of N-acetylglucosamine is bonded adjacent to galactose, and this bond is a β-glycoside bond, and the binding position is at the 3-position or 4-position. It is preferable that And if it has these structures, the sugar chain etc. may couple | bond with the glucosamine residue next. Among these, examples of the receptor pseudo-sialoglycosaccharide in the present invention include a Neu5Acα2-6Galβ1-4 (3) GlcNAcβ1-structure and a Neu5Acα2-3Galβ1-4 (3) GlcNAcβ1-structure. Neu5Acα2-3Gal is a structure in which N-acetylneuraminic acid (Neu5Ac) is α2-3 linked to galactose (Gal). Human parainfluenza virus type 1 (hPIV-1) and type 3 (hPIV-3) ) Is known to bind to a sugar chain having Neu5Acα2-3Gal.

  Furthermore, since the receptor pseudo-sialog sugar chain moiety of the compound of the present invention is bonded to the hydrophobic moiety and the polymer moiety via the linker moiety shown below, it has a reactive functional group at its end. The functional group in this case may be any functional group that can be bonded to the functional group present in the linker portion by chemical reaction, for example, amino group, azide group, carboxyl group, acetylene group, thiol group, aldehyde group, hydroxy group. , Α, β-unsaturated esters and α, β-unsaturated carbonyl groups including α, β-unsaturated amides. The hemagglutinin binding ability of the receptor pseudo-sialog sugar chain moiety in the present invention is detected by binding the receptor pseudo-sialog sugar chain or influenza virus or hemagglutinin protein to a carrier as appropriate, adding the other, binding, and washing. Examples of such detection methods include conventional methods such as ELISA, Western blotting, and immunofluorescent staining, as well as methods for detecting influenza virus by measuring sialidase activity.

  The “polymer moiety” in the compound of the present invention is a polymer that is not toxic to the living body or low in toxicity, and it is sufficient if it can exert a spatial inhibitory effect when the compound of the present invention is bound to the surface of influenza virus. Preferably, it is 20 angstroms or more, more preferably 50 angstroms or more, still more preferably 80 angstroms or more, and most preferably the chain length is 100 angstroms or more. Examples of the polymer that is not toxic to a living body include polymers such as polyglutamic acid, polyaspartic acid, hydroxyethyl acrylamide, and styrene-maleic acid copolymer. A heteropolymer in which plural kinds of monomers of at least one kind are appropriately bonded may be used, and the composition, bonding order, etc. thereof are not particularly limited. The molecular weight of the “polymer moiety” is not particularly limited as long as the compound of the present invention can exert a spatial inhibitory effect when bound to the surface of influenza virus. For example, the number average molecular weight is in the range of 1,000 to 150,000. Preferably within the range of 30,000 to 100,000, more preferably within the range of 45,000 to 85,000, and most preferably 55,000 to 70,000. Range. As the “polymer portion”, polyglutamic acid and hydroxyethylacrylamide can be suitably exemplified. Among them, the polymerization degree is preferably 50 to 1000, more preferably the polymerization degree is 300 to 800, and still more preferably the polymerization degree is 400 to 700, Most preferred examples include polyglutamic acid having a polymerization degree of 500 to 600 and polyglutamic acid having a polymerization degree of about 540. Here, the polyglutamic acid is preferably α-polyglutamic acid. In addition, such a polymer portion may have a functional group for bonding by a chemical reaction or the like with a functional group of a linker portion that binds the above-mentioned receptor pseudo-sialog sugar chain portion having hemagglutinin binding ability and a hydrophobic portion. The number of such functional groups is preferably 1% or more of the constituting unit structure. For example, the following functional group, amino group, azide group, carboxyl group, acetylene group, thiol group, aldehyde group, hydroxy group, α, Examples include α, β-unsaturated carbonyl groups, including β-unsaturated esters and α, β-unsaturated amides, and the like, in order to couple the polymer moiety of the present invention with other components of the present invention. Is preferably a terminal-modified polymer, specifically a polymer moiety that is an N-terminal acetylated polyglutamic acid. Further, such a polymer portion may be subjected to sugar chain modification, preferably 1 to 50%, more preferably 2 to 30%, still more preferably 3 to 10%, and especially a polymer portion having a sugar chain introduction rate of 5%. The example which uses can be illustrated suitably.

  Moreover, the polymer which comprises a coating material, a synthetic fiber, etc. can be utilized as said "polymer part." In this case, the molecular weight of the “polymer moiety” is not particularly limited as long as the receptor pseudo-sialoglycosaccharide moiety of the compound of the present invention can adsorb influenza virus. For example, the number average molecular weight is 1,000 to 150,000. It is preferably within the range, and more preferably within the range of 2,000 to 100,000. Further, the ratio of the compound of the present invention in the polymer compound is 1% to 50%, preferably 2% to 30%, more preferably 3% to 10% in terms of molar ratio.

  The “linker moiety” in the compound of the present invention may be any as long as it plays the role of connecting the above-described receptor pseudo-sialoglycosyl moiety having hemagglutinin binding ability, the hydrophobic moiety described below, and the polymer moiety, What is necessary is just to have at least three reaction points in order to combine these three. The length of the linker moiety is preferably 1 angstrom or more, more preferably 3 angstrom or more, and even more preferably 5 angstrom or more, and each of the three members is bonded by a functional group or the like by a functional group. Functional groups include amino, azide, carboxyl, acetylene, thiol, aldehyde, hydroxy, α, β-unsaturated esters and α, β-unsaturated amides including α, β-unsaturated amides. A group etc. can be illustrated and what is necessary is just a combination which can be couple | bonded appropriately with each structural part by chemical reaction. Specifically, the linker moiety of the present invention includes lysine, ornithine, 2,6-diaminohexanoic acid, 3,5-diaminobenzoic acid, 3,4-diaminobenzoic acid, 1,2-diaminohexanecarboxylic acid and the like. Among them, for example, lysine, ornithine and the like can be preferably used as the amino acid residue of the trifunctional amino acid.

  The “hydrophobic moiety” in the compound of the present invention is not particularly limited as long as the binding between the compounds of the present invention and the compound of the present invention and influenza virus is enhanced by hydrophobic interaction. Examples thereof include a compound, an alkyl group that may have a branched structure or an unsaturated bond, and cholesterol, cholestanoic acid, cholic acid, or C1-50, more preferably C1-40, Preferably, an alkyl group having 3 to 35 carbon atoms can be suitably exemplified. The number and position of branched structures and unsaturated bonds are not particularly limited, and such alkyl groups are multifunctional including other molecules such as glycerol, hydroxyacetic acid, hydroxypropionic acid, ethylene glycol, ethanolamine, amino acids. It may be bonded to a compound or the like. Examples of the alkyl group include methyl, ethyl, propyl, isopropyl, butyl, secondary butyl, tertiary butyl, isobutyl, amyl, isoamyl, tertiary amyl, pentyl, hexyl, heptyl, 2-heptyl, Isoheptyl, tertiary heptyl, n-octyl, isooctyl, tertiary octyl, 2-ethylhexyl, nonyl, isononyl, decyl, undecyl, dodecyl, tridecyl, tetradecyl, pentadecyl, hexadecyl, heptadecyl, octadecyl, nonadecyl, eicosyl, behenyl, Examples thereof include linear or branched alkyl groups such as tricosyl. In addition, the hydrophobic portion has a functional group for bonding with a functional group of the linker portion by chemical reaction, etc., as the functional group, for example, amino group, azido group, carboxyl group, acetylene group, thiol group, aldehyde group, Examples thereof include an α, β-unsaturated carbonyl group including a hydroxy group, an α, β-unsaturated ester and an α, β-unsaturated amide. Among them, a carboxyl group that preferably forms an amide bond with an amino group can be mentioned.

  The compound of the present invention can be synthesized by sequentially combining these materials using, as materials, a receptor pseudo-sialog sugar chain portion having an independent hemagglutinin binding ability, a linker portion, a hydrophobic portion, and a polymer portion. It is also possible to synthesize by combining partial compounds synthesized in advance by integrating a plurality of parts, or by combining a partial compound and another part. In addition, each part may be synthesized as one molecule or may be synthesized by combining a plurality of molecules. For example, each part may be formed only after completion. Any compound may be used as long as the receptor pseudo-sialog sugar chain portion having hemagglutinin binding ability, the hydrophobic portion, and the polymer portion are bonded via the linker portion. For example, as shown in FIG. 2, the compound of the present invention can be synthesized from the three compounds of blocks 2, 11, and 18 as the target 1 compound, or the four compounds of blocks 2, 11, 18, and 5 can be synthesized. From the above, the compound of the present invention can also be synthesized as a target 2 compound.

  The compounds of the present invention may be prepared by standard chemical methods well known to those skilled in the art. The binding of each component is appropriately selected depending on the type of functional group possessed by each element, and can include steps such as protection and deprotection of the functional group as appropriate. Hereinafter, a general method for synthesizing the compound of the present invention is exemplified, but the method for synthesizing the compound of the present invention is not limited thereto. Each site in the compound of the present invention is a receptor pseudo-sialoglycosaccharide moiety represented by the formula (IV):

A hydrophobic moiety of formula (V),

A polymer moiety represented by formula (VI),

A linker moiety of formula (VII),

Each of the binding functional groups R ¹ , R ² , R ³ , R ^1a , R ^2a, and R ^3a is a bond of R ¹ -R ^1a , R ² -R ^2a , R ³ -R ^3a . Is a group capable of forming Further, the reactive functional ^{R 1} receptor pseudo sialo carbohydrate moieties, reactive functional ^{R 2} hydrophobic moieties, reactive functional ^{R 3} polymer moieties, reactive functional group ^R 1a in the linker ^{portion, R 2a} and ^{R 3a} is For example, the binding functional group shown in Table 1 can be used to form a bond of R ¹ -R ^1a , R ² -R ^2a , R ³ -R ^3a shown in Table 1. R ¹ , R ² , R ³ , R ^1a , R ^2a and R ^3a can be a combination in which R ¹ and R ^1a , R ² and R ^2a , and R ³ and R ^3a can form the bond shown in Table 1. For example, the same functional group may be used at a plurality of locations, or all different functional groups may be used. When the functional groups R ¹ , R ² , R ³ , R ^1a , R ^2a and R ^3a involved in the reaction are amino groups (—NH ₂ ), these are carboxyl groups (—COOH) or aldehyde groups (—CHO) and It can react to form an amide or imine.

  Examples of amino-protecting groups include carbamates represented by methyl carbamate, ethyl carbamate, 9-fluorenylmethyl carbamate (Fmoc) and Fmoc derivatives, and phthalimides such as phthalimide and 4-nitrophthalimide. However, Fmoc is preferable from the viewpoint of easy introduction of a protecting group and easy removal. The azide group can be easily converted to the corresponding amino group by reduction using a Staudinger reaction using a phosphine compound such as triphenylphosphine as a reducing agent or a mild hydride such as sodium hydroborate. Therefore, an azide group can also be used as a precursor of an amino group.

  As a method for forming an amide, a site having an amino group, a site having a carboxyl group, and an activator of a carboxyl group such as N-hydroxysuccinimide, N-hydroxyphthalimide, 1-hydroxybenzotriazole, or the like can be used. -The target amide is formed by dissolving in an organic solvent such as dimethylformamide and reacting with a dehydrating agent such as dicyclohexylcarbodiimide, 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide or its hydrochloride. A method can be mentioned. In addition to this, an amide is formed by reacting the amine with an amine in an organic solvent after previously converting the carboxyl group into an active ester with N-hydroxysuccinimide, N-hydroxyphthalimide, 1-hydroxybenzotriazole, or the like. It can also be done.

  As a method for forming imine, a dehydration condensation reaction is performed by dissolving a site having an amino group and a site having a carboxyl group in an organic solvent such as dichloromethane or toluene and coexisting a dehydrating agent such as sodium sulfate, magnesium sulfate or alumina. And forming a target imine. In addition to this, the amino group-containing moiety and the carboxyl group-containing moiety are dissolved in an aromatic solvent such as benzene or toluene to catalyze a non-volatile acid such as benzenesulfonic acid, toluenesulfonic acid, or camphorsulfonic acid. The target imine can be formed by dehydration condensation using a Dean-Stark reactor.

When any of the functional groups R ¹ , R ² , R ³ , R ^1a , R ^2a or R ^3a involved in the reaction is an azide group (—N ₃ ), these react with the acetylene group, and 1, 2, 3 -Triazoles can be formed. Moreover, since an azide group is a stable functional group, it usually does not require a protecting group.

  As the formation reaction of 1,2,3-triazole, these functional groups are added by 1,3-dipolar cycloaddition by dissolving a site having an azide group and a site having an acetylene group in an organic solvent or water. A method of forming the desired 1,2,3-triazole by reacting (Fisgen reaction) can be mentioned. This reaction proceeds even without a catalyst, but the reaction is completed quickly by using monovalent copper ions as a catalyst (click chemistry).

When any of the functional groups R ¹ , R ² , R ³ , R ^1a , R ^2a or R ^3a involved in the reaction is a carboxyl group (—COOH), these are an amino group (—NH ₂ ) or a hydroxyl group (— OH) to form amides or esters.
Examples of the carboxyl-protecting group include methyl ester, 9-fluorenyl methyl ester, methoxymethyl ester, methylthiomethyl ester, benzyloxymethyl ester, trichloroethyl ester, trimethylsilylethyl ester, tetrahydrofuranyl ester, tert-butyl ester and the like. Examples include esters and silyl esters such as tert-butyldimethylsilyl ester, tert-butyldiphenylsilyl ester, and triisopropylsilyl ester. The method for forming the amide is as described above.
As a method for forming an ester, a site having a carboxyl group and a site having a hydroxyl group are dissolved in an organic solvent such as dichloromethane or N, N-dimethylformamide, and a carboxyl group activator and 4-N as a catalyst are used. By carrying out the reaction in the presence of, N-dimethylaminopyridine, there can be mentioned a reaction for forming the target ester. Examples of the carboxyl group activator include carbodiimides such as dicyclohexylcarbodiimide, 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide and its hydrochloride, 2,4,6-trichlorobenzoyl chloride (Yamaguchi method), 1 Activators such as -methyl-6-nitrobenzoic acid (Shiina method) and 2-halo-N-alkylpyridinium salts (Mukayama condensation reagent) can be used, and an organic base such as tributylamine is used as necessary. It may be used.

When any of the functional groups R ¹ , R ² , R ³ , R ^1a , R ^2a or R ^3a involved in the reaction is a thiol group (—SH), these react with the thiol group (—SH) of the binding partner. And disulfide bonds can be formed. The thiol group forms a sulfide by a Michael reaction with an α, β-unsaturated carbonyl group (—COCH ₂ CH═CH ₂ ) including an α, β-unsaturated ester and an α, β-unsaturated amide. I can do it. Since the thiol group is a stable functional group, it usually does not require a protecting group.

  As a method for forming a disulfide bond, a site having two thiol groups may be mixed in a solvent and oxidized to form a disulfide bond, but in this case, formation of an undesired homodimer cannot be avoided. . Therefore, after activating a compound having one thiol group with 1-chlorobenzotriazole, the compound having the other thiol group and benzotriazole are added to the reaction solution to selectively form a desired disulfide bond. be able to. At this time, the reaction can be efficiently advanced by adding an excessive amount of thiourea.

  The sulfide is formed by reacting a site having a thiol group and a site having an α, β-unsaturated carbonyl group in an organic solvent in the presence of a Lewis acid catalyst to form a desired sulfide bond. be able to.

When any of the functional groups R ¹ , R ² , R ³ , R ^1a , R ^2a or R ^3a involved in the reaction is a hydroxyl group (—OH), these react with a carboxyl group (—COOH) to form an ester Can be formed.

  Protecting groups for hydroxyl groups include methoxymethyl ether, methylthiomethyl ether, 4-methoxybenzylmethyl ether, o-nitrobenzyloxymethyl ether, trichloroethoxymethyl ether, tetrahydropyranyl ether, allyl ether, benzyl ether, 4-methoxybenzyl Ethers such as ether, trimethylsilyl ether, triethylsilyl ether, triisopropylsilyl ether, silyl ethers such as tert-butyldimethylsilyl ester, tert-butyldiphenylsilyl ester, esters such as acetyl ester and pivaloyl ester, 9- Fluorenyl methyl ester carbonate, vinyl carbonate, 2- (trimethylsilyl) ethyl carbonate, 2,2,2-trichloro Ethylcarbonate, carbonic esters such as tert- butyl carbonate can be given. The method for forming the ester is as described above.

When any of the functional groups R ¹ , R ² , R ³ , R ^1a , R ^2a or R ^3a involved in the reaction is an aldehyde group (—CHO), these react with an amino group (—NH ₂ ). Can form imines. Since the aldehyde group is usually stable, no protective group is required, but if a protective group is used, 1,2-ethanediol, 1,3-propanediol, 2-trimethylsilyl-1,3-propanediol There is a method of leading to the corresponding acetal using diols such as o-catechol. The method for forming imine is as described above.

When any of the functional groups R ¹ , R ² , R ³ , R ^1a , R ^2a or R ^3a involved in the reaction is an acetylene group, these react with an azide group (—N ₃ ), and 1, 2, 3-triazole can be formed. Acetylene groups are usually stable and do not require a protecting group. The method for forming 1,2,3-triazole is as described above.

Α, β-unsaturation in which any of the functional groups R ¹ , R ² , R ³ , R ^1a , R ^2a or R ^3a involved in the reaction comprises an α, β-unsaturated ester and an α, β-unsaturated amide When they are carbonyl groups (—COCH ₂ CH═CH ₂ ), they can react with thiol groups (—SH) to form sulfides. The α, β-unsaturated carbonyl group is usually stable and does not require a protecting group. The method for forming sulfide is as described above.

  Examples of the compound of the present invention include the following formula (II).

  In the formula, PGA represents the formula (III)

M and n that satisfy (m + 1) n = 50 to 1000 are desirable. m and n are integers. These can be synthesized by the following methods and the methods described in Examples.

  Moreover, the compound of this invention is also compoundable with the following method. For example, the coupling of block 2 and block 11 in FIG. 2 below involves blocking the carboxyl group of block 11 to an active ester with N-hydroxysuccinimide, N-hydroxyphthalimide, 1-hydroxybenzotriazole, etc. By reacting with 2, an amide derivative (block [2 + 11]) composed of block 2 and block 11 can be obtained. Formula (VIII) shows the coupling of block 2 and block 11 using N-hydroxysuccinimide.

  The block [2 + 11] is obtained by dissolving the block 2, the block 11 and 1-hydroxybenzotriazole in a solvent such as dichloromethane, adding a dehydrating agent such as dicyclohexylcarbodiimide, and dehydrating and condensing them. You can also.

  In this block [2 + 11], Nω derived from lysine is protected with Fmoc, so that the de-Fmoc form of block [2 + 11] can be obtained by removing this Fmoc group with an amine. The amine used in this reaction is not particularly limited, and preferred examples include triethylamine, piperidine, piperazine and morpholine. The de-Fmoc form of the block [2 + 11] thus obtained was obtained by converting sodium polyglutamate corresponding to block 18 to 4- (4,6-Dimethoxy-1,3,5-triazin-2-yl) -4-methylmorpholinium Chloride (DMTMM). ) Can be efficiently reacted, and the target compound (block [2 + 11 + 18]) can be obtained as shown in formula (IX). At this time, by changing the mixing ratio of the block [2 + 11] and the block 18 in the reaction, compounds having different sugar chain introduction rates can be synthesized.

  The coupling between the block [2 + 11] and the block 18 is not limited to the above example. For example, the de-Fmoc form of the block [2 + 11] and the carboxyl group are previously activated with N-hydroxysuccinimide. The target compound (block [2 + 11 + 18]) can be obtained by reacting the present block 18. Also, the de-Fmoc form of block [2 + 11] and the polymer corresponding to block 18 not activated with N-hydroxysuccinimide are dissolved in a solvent such as dichloromethane, and a dehydrating agent such as dicyclohexylcarbodiimide is added thereto. The block [2 + 11 + 18] can also be obtained by dehydrating and condensing them.

  In addition, examples of the compound of the present invention include a compound having a spacer containing a polyether between a linker moiety and a sugar chain moiety represented by the following formula (X), which can be synthesized by the following method. it can.

  In the formula, PGA represents the formula (III)

M and n are integers, and (m + 1) n is preferably 50 to 1000, more preferably 300 to 800, more preferably 400 to 700, and most preferably 500 to 600. it can. Especially, the compound whose (m + 1) n is about 560 can be illustrated suitably, and it can synthesize | combine with the following method.

  The coupling between block 5 and block 11 in FIG. 2 includes the Staudinger reaction using the azide group of block 5 as a reducing agent (a) a phosphine compound such as triphenylphosphine, and (b) mild such as sodium hydroborate. Reduction to the corresponding amine by reduction with a simple hydride. By dissolving this amine, block 11 and 1-hydroxybenzotriazole in a solvent such as dichloromethane, adding a dehydrating agent such as dicyclohexylcarbodiimide, and dehydrating and condensing them, as shown in formula (XI), An amide derivative (block [5 + 11]) composed of block 5 and block 11 can be obtained.

  As described above, the compound in which the carboxyl group of block 11 is led to an active ester with N-hydroxysuccinimide, N-hydroxyphthalimide, 1-hydroxybenzotriazole, etc. is reacted with the amine obtained by reducing block 5 Block [5 + 11] can also be obtained.

  In addition, since the carboxyl group of the block [5 + 11] is a methyl ester, it is selectively hydrolyzed using a base such as lithium hydroxide, sodium hydroxide, or potassium hydroxide, and the block [5 + 11] A demethylated form can be obtained. In this case, suitable conditions include using a lithium hydroxide aqueous solution as a base and reacting in THF at a low temperature. By dissolving the demethylated form of block [5 + 11] thus obtained, block 2 and 1-hydroxybenzotriazole in a solvent such as dichloromethane, adding a dehydrating agent such as dicyclohexylcarbodiimide, and dehydrating and condensing them. As shown in formula (XII), an amide derivative (block [2 + 5 + 11]) composed of block 2 and block [5 + 11] can be obtained.

  This Nω in block [2 + 5 + 11] removes the Fmoc group with an amine as described above, and sodium polyglutamate corresponding to block 18 is 4- (4,6-Dimethoxy-1,3,5-triazin-2- (yl) -4-methylmorpholinium Chloride (DMTMM) can be used to react efficiently, and the target compound (block [2 + 5 + 11 + 18]) can be obtained as shown in formula (XIII). Similarly, compounds with different sugar chain introduction rates can be synthesized by changing the mixing ratio of the reactants.

  The salt of the compound of the present invention may be non-toxic when administered to a living body, and examples thereof include acid addition salts, metal salts, ammonium salts, organic amine addition salts, etc. Examples of acid addition salts include hydrochloric acid, sulfuric acid, As each inorganic acid salt and metal salt such as nitric acid and phosphoric acid, each alkali metal salt such as lithium, sodium and potassium, each alkaline earth metal salt such as magnesium and calcium, each metal salt such as aluminum and zinc, Examples of ammonium salts include ammonium and tetramethylammonium salts, and examples of organic amine salts include triethylamine, piperidine, morpholine, toluidine, and the like, and exist as hydrates and solvates. You may do it. In the compound of the present invention, a hydroxyl group, a carboxyl group, an amino group and the like may be protected with an appropriate protecting group. Such protecting groups are not particularly limited, and any protecting group may be used as long as it can be used by those skilled in the art. Furthermore, the compounds of the present invention may exist as pure forms of isomers such as optically active forms and diastereoisomers, or any mixture thereof (for example, racemates and diastereoisomer mixtures). Any of the materials described herein are within the scope of the present invention.

  The compound of the present invention or a salt thereof may have a fluorescent functional group so that it can visually follow the in vivo and intracellular movement, intracellular movement or localization of the virus after binding to the virus. . Such a fluorescent functional group may be added to any part, but for example, it is preferably introduced into a spacer part. The type of the fluorescent functional group is not particularly limited. For example, BODIPY group (4,4-difluoro-5,7-dimethyl-4-bora-3α, 4α-diaza-s-indacene-3-propionic acid), rhodamine , Cy3, Cy5, FITC, or the like can be used.

The compound of the present invention or a salt thereof is a compound or a salt thereof, which is composed of a receptor pseudo-sialog sugar chain moiety having an HA binding ability, a hydrophobic moiety, and a polymer moiety bound via a linker moiety, Influenza virus can be effectively inhibited by 3 steps (FIG. 14).
Step 1) Inhibition of HA function: the receptor pseudo-sialoglycosaccharide moiety having the ability to bind HA specifically binds to the HA receptor binding pocket;
Step 2) Enhanced binding: binding of influenza virus to the compound of the invention by hydrophobic interaction;
Step 3) Spatial inhibition: the polymer moiety surrounds and inhibits viral particles such as NA spikes by hydrophobic interactions;

  Thus, the compound of the present invention or a salt thereof is a natural common to all influenza A viruses such as hemagglutinin subtype (H1-H16) and neuraminidase subtype (N1-N9), and all influenza B viruses. It is a pseudo-molecule of the sialoglycan receptor that is a receptor. Therefore, it is not only capable of adsorbing all types of A and B influenza viruses that have been prevalent in the past and in the past, but also strong against all new types of influenza A and B influenza viruses that may be prevalent in the future. Has adsorption capacity. Further, the compound of the present invention or a salt thereof not only binds to viral hemagglutinin (HA) but also clings to the surface of the virus, thereby strongly inhibiting sialidase activity of neuraminidase (NA) spike in addition to inhibition of hemagglutinin spike function. (FIG. 14). By inhibiting HA and NA, it is possible to inhibit multiple mechanisms such as adsorption of the virus to the cell surface, virus budding, and virus infection. Since it is extremely rare that mutations into HA and NA occur at the same time, it can be said that the compound of the present invention that inhibits HA and NA is a compound designed so that a resistant strain is hardly formed. Therefore, it is considered that the compound of the present invention has an effect on influenza mutants.

  In addition, even if the receptor pseudo-sialoglycosyl moiety of the compound of the present invention is cleaved by sialic acid by influenza virus sialidase, the exposed galactose residue is captured by the host macrophage and the virus itself is phagocytosed by the macrophage. Therefore, growth in the host can be suppressed.

  Therefore, the compound of the present invention or a salt thereof is an influenza virus hemagglutinin function inhibitor, an influenza virus sialidase inhibitor, an influenza virus hemagglutinin function and sialidase inhibitor, an influenza virus infection inhibitor, or an influenza virus cell surface inhibitor. Adsorption inhibitors, influenza virus budding inhibitors, influenza virus infection, adsorption and budding inhibitors (hereinafter also referred to as "inhibitors of the present invention"), influenza preventive and / or therapeutic agents ( Hereinafter, it may be referred to as “the preventive or therapeutic agent of the present invention”. Since the compound of the present invention or a salt thereof can simultaneously inhibit HA function and NA, it can be suitably used as an influenza virus hemagglutinin function and sialidase inhibitor. In addition, the compound of the present invention has the effect of the above three steps, and can inhibit any step of infection, adsorption and budding of influenza virus, and thus has an effect on both prevention and treatment of influenza virus. . Therefore, the compound of the present invention or a salt thereof can be suitably used as an influenza preventive agent, an influenza therapeutic agent, an influenza preventive and therapeutic agent, that is, an influenza preventive and / or therapeutic agent. The compound of the present invention or a salt thereof can inhibit the adsorption, budding, and infection of a virus to a host cell by inhibiting HA function and NA, and the conventional anti-influenza drugs Tamiflu and Relenza which are NA inhibitors More effectively, influenza can be prevented and / or treated.

  In another aspect, the present invention provides a method of using the compound of the present invention or a salt thereof as the inhibitor of the present invention, an influenza preventive and / or therapeutic agent, or the present invention for inhibiting influenza virus hemagglutinin. Inhibiting infection of influenza virus compounds or salts thereof, compounds of the present invention or salts thereof for inhibiting influenza virus sialidase, compounds of the present invention or salts thereof for inhibiting influenza virus hemagglutinin and sialidase The compound of the present invention or a salt thereof, the compound of the present invention for inhibiting adsorption of influenza virus or a salt thereof, the compound of the present invention for inhibiting budding of influenza virus or a salt thereof, The present invention for inhibiting infection, adsorption and budding Compound or and its salts, preventing influenza, treat influenza, or compounds of the present invention for preventing and treating influenza or its salt or the like including.

  The subject of application of the compound of the present invention or a salt thereof, the preventive or therapeutic agent for influenza of the present invention, or the inhibitor of the present invention is a virus having a receptor protein that binds to a receptor pseudo-sialog sugar chain moiety having HA binding ability. Well, an influenza virus can be illustrated suitably. As influenza virus, any of A type, B type, and C type may be sufficient, A type and B type can be illustrated suitably and HA type and NA type of influenza virus are not particularly limited. The target virus may be any virus that can infect humans, and may be a virus that has the ability to infect pigs and birds. Subjects to be administered may be infected with, or expected to be infected with, an influenza virus capable of infecting humans such as humans, monkeys, pigs, dogs, horses, sheep, cats , Rabbits, rats, mice, hamsters, ferrets, etc., and preferably humans. In addition, the inhibitor of the present invention can be administered to a biological sample such as a body fluid, tissue or cell isolated from the individual mentioned as the administration subject. Anything that can be recognized by the above-mentioned receptor pseudo-sialog sugar chain moiety can also be used for microbial infections such as viruses and bacteria. For this purpose, the receptor pseudo-sialog sugar chain moiety can be appropriately selected.

  As the influenza preventive or therapeutic agent of the present invention or the inhibitor of the present invention, the compound of the present invention or a physiologically acceptable salt thereof may be used alone, but pharmacologically and pharmaceutically acceptable. Preferably, the pharmaceutical composition containing the compound of the present invention or a salt thereof as an active ingredient is used using an additive / carrier for pharmaceutical preparations. The route of administration of the influenza preventive or therapeutic agent of the present invention or the inhibitor of the present invention to a living body is not particularly limited, and in addition to oral administration, injections, instillations, suppositories, inhalants, transmucosal absorbents, transmucosal agents, It can also be administered parenterally using skin absorbents, nasal drops, ear drops and the like. In the case of oral administration, the influenza preventive or therapeutic agent of the present invention can be prepared as a preparation for oral administration such as a tablet, capsule, fine granule, granule, liquid, or syrup. Furthermore, when administering the inhibitor of this invention to biological samples, such as a bodily fluid, a tissue, a cell, it can add directly to a biological sample, and can also add to a cell culture solution, a tissue preservation solution, etc. Such a biological sample may be added with a preservative, a buffer, a pH adjuster and the like. The dosage form of the influenza preventive or therapeutic agent of the present invention and the inhibitor of the present invention is not particularly limited, and can be appropriately selected from storage stability and ease of operation, and examples thereof include powders, tablets, and liquids. . In the case of a powder or tablet, it can be appropriately dissolved in a solvent to prepare a liquid.

  Examples of the pharmacologically and pharmaceutically acceptable additives and carriers for the influenza preventive or therapeutic agent of the present invention and the inhibitor of the present invention include various organic or inorganic substances commonly used as pharmaceutical materials. For example, excipients, lubricants, pH adjusters, binders, disintegrants, solvents, solubilizers, suspending agents, isotonic agents, buffers, soothing agents, absorption enhancers, etc. A base material is mentioned. Further, water, an aqueous solution, or a water-miscible liquid such as glycerin or polyethylene glycol can be used. The dosage of the influenza preventive or therapeutic agent of the present invention and the inhibitor of the present invention depends on various conditions such as the health status of the individual to be administered, symptoms, presence or absence of other complications, constitution, age, purpose of prevention or treatment, etc. The dosage can be appropriately increased or decreased depending on the dosage, the dosage can be divided into 1 to several times, and the administration period can be appropriately selected according to the symptom and the like. The dose of the influenza preventive or therapeutic agent of the present invention is 0.001 μg / kg body weight to 1 g / kg body weight, preferably 0.01 μg / kg body weight to 100 mg / kg body weight, more preferably 0.1 μg per day. / Kg body weight to 10 mg / kg body weight can be exemplified. In addition, the dose, the number of administrations, and the administration period of the influenza preventive or therapeutic agent of the present invention and the inhibitor of the present invention to a biological sample can be appropriately adjusted, and 0.1 pM to 1 mM, preferably 1 pM per day. -100 μM, more preferably 0.1 nM to 10 μM can be exemplified.

  Other aspects of the present invention include an adsorbent for influenza virus containing the compound of the present invention or a salt thereof as an active ingredient, and an adsorbent for influenza virus carrying the compound of the present invention or a salt thereof. The adsorbent for influenza virus of the present invention is usually used as a solution type adsorbent used alone, or as a raw material for the adsorbent for influenza virus of the present invention. As a medium for preparing the solution type adsorbent, any medium can be used as long as the compound of the present invention or a salt thereof is dissolved. Specifically, water, lower alcohol, ethylene glycol, or a mixture thereof can be used. A solution. Among these, it is preferable to use a mixed solution of water and ethanol or a mixed solution of water and isopropanol as a medium. The concentration of the compound of the present invention or a salt thereof in the solution is not particularly limited as long as influenza virus can be adsorbed, but is, for example, 0.00001 to 0.01% by weight, preferably 0.00005 to 0.005% by weight. Preferably it is 0.0001 to 0.001% by weight. The solution for adsorbing influenza virus can contain a dispersant, a fragrance, a coloring agent, an antioxidant, and the like, if necessary.

  As a method of using the solution for adsorbing influenza virus, spray this solution with a spray or the like to adsorb and remove influenza virus floating in the air, or bubbling air containing influenza virus into this solution to remove influenza virus. A method of taking out the removed clean air can be exemplified.

  An influenza virus adsorbent in which the compound of the present invention or a salt thereof is supported on a base can also be produced using this influenza virus adsorption solution. For example, by immersing a base such as cloth, paper, or wood in this solution, the base such as cloth, paper, or wood is used as an adsorbent for influenza virus carrying the compound of the present invention or a salt thereof. Can do. Using the influenza virus adsorbent thus obtained as a raw material, products such as filters, masks, towels, fabrics, curtains, wallpaper, and building materials can be produced. On the other hand, the compound of the present invention is coated on a substrate that cannot be impregnated with a solution for adsorbing influenza virus, such as a plastic, metal, glass, etc. by spraying, coating, or the like. Alternatively, a salt thereof can be supported. Since the compound of the present invention has a polymer portion, it can be easily supported because it electrostatically interacts with the surface of the substrate.

  In addition, an adsorbent for influenza virus in which the compound of the present invention or a salt thereof is supported on a polymer compound can also be produced. Such support forms include support by the covalent bond between the compound of the present invention or a salt thereof and the polymer compound, or the surface of the polymer compound and the polymer part of the compound of the present invention or the salt thereof non-covalently interact with each other. A carrier to be attached by acting can be mentioned. As such a polymer compound, any polymer compound can be used. For example, acrylic acid ester, acrylic-styrene copolymer, acrylic-vinyl acetate copolymer, ethylene-vinyl acetate copolymer Specific examples include acrylic-silicone-acrylic resin-based, ethylene-vinyl chloride copolymer, styrene-butadiene copolymer, and epoxy-based, urethane-based, melamine-based polymers, and the like. Those skilled in the art can appropriately select a preferable polymer compound according to the intended use state and use state.

  In the case of a supported form by covalent bonding between the compound of the present invention or a salt thereof and the polymer compound, as described above, such a polymer compound (polymer) can be used as the “polymer portion” of the compound of the present invention. Covalent bonding of the compound of the present invention or a salt thereof and the polymer compound can be performed, for example, by polymerizing the monomer constituting the polymer compound and the polymer portion of the compound of the present invention or salt thereof. . In addition, it is also possible to covalently bond the reactive functional group on the surface of the polymer compound by reacting the compound of the present invention or a salt thereof.

  The adsorbent for influenza virus in which the compound of the present invention or a salt thereof is supported on a polymer compound can be used as, for example, paints, plastic materials, synthetic fibers and the like. For example, when used as a paint, the coating film functions as an adsorbent for influenza virus. When used as a plastic material, the surface functions as an adsorbent for influenza virus. When used as a synthetic fiber, it is used as a filter, a mask, a towel, a curtain or the like having an influenza virus adsorption action.

  In addition, the compound of the present invention or a salt thereof has a pseudo-sialog sugar chain portion of a human receptor for influenza virus. Therefore, it can also be used as a device (kit) for monitoring that the avian influenza virus has acquired the binding to the human receptor by mutation. In addition, it can also be used as a material for selectively adsorbing, separating and purifying only viruses and microorganisms having a binding property to the sialosaccharide chain of human-type receptor from various virus mixtures. Furthermore, it can be used as a material for investigating in vivo, intracellular movement and localization of the compound of the present invention or a salt thereof bound to influenza virus by binding to a fluorescent molecule or polymer.

  The following examples further illustrate the present invention, but the scope of the present invention is not limited by the examples.

  The compound of the present invention, Neu5Acα (2-6) Galβ (1-4) GlcNAcon PGA (sugar chain introduction rate: 5%) was produced by the following method. A synthesis scheme is shown below.

[Synthesis of Compound 3]
Compound 1 (450 mg, 0.627 mmol / manufactured by Tokyo Chemical Industry) and compound 2 (750 mg, 0.987 mmol / manufactured by Tokyo Chemical Industry) were dissolved in DMF / MeOH (1/1, 60 ml) and stirred for 18 hours. The completion of the reaction was confirmed by TLC (AcOEt / MeOH / H ₂ O / AcOH = 4/2/1 / 0.5) and then concentrated under reduced pressure. The obtained residue was subjected to column chromatography (CHCl ₃ / MeOH = 3/1 → 2/1 → 1/1) to obtain the compound 3 (273 mg, 32%).
Rf value of Compound 3: 0.63 (AcOEt / MeOH / H2O / AcOH = 4/2/1 / 0.5) According to HPTLCplates, Silica gel 60 F254. The NMR spectrum of Compound 3 is shown in FIG.

[Synthesis of Compound 4]
Compound 3 (260 mg, 0.191 mmol) was dissolved in MeOH (12 ml), Et ₃ N (0.6 ml) was added, and the mixture was stirred for 20 hours. The completion of the reaction was confirmed by TLC (AcOEt / MeOH / H ₂ O / AcOH = 4/2/1 / 0.5) and then concentrated under reduced pressure. The obtained residue was subjected to gel filtration column chromatography (LH-20, MeOH) to obtain Compound 4 (211, mg, 97%).

[Synthesis of Compound 5]
Compound 4 (158 mg, 0.139 mmol) and polyglutamate Na (average molecular weight 84,600, 419 mg, 2.77 mmol (as Na glutamate)) were dissolved in H ₂ O (100 ml), THF (50 ml) and DMTMM: 4 -(4,6-Dimethoxy-1,3,5-triazin-2-yl) -4-methylmorpholinium Chloride (385 mg, 1.39 mmol) was added and stirred for 2 hours. After completion of the reaction was confirmed by TLC (AcOEt / MeOH / H ₂ O / AcOH = 4/2/1 / 0.5), EtOH was added and the mixture was concentrated under reduced pressure. The obtained residue was dissolved in 5% NaHCO ₃ aqueous solution (80 ml) and subjected to gel filtration column chromatography (G-25, MeOH) to obtain compound 5 (530 mg, 92%). A polymeric compound containing a sugar chain (hereinafter, also referred to as “polyglutamic acid compound having a sugar chain introduction rate of 5%”) was obtained.
In a similar manner, 0.04 mmol of compound 4 was used to give a polymeric compound containing 1% of the sugar chain of the present invention, and 0.28 mmol of compound 4 was used to obtain 10% of the sugar chain of the present invention. A polymeric compound was obtained.

The polyglutamic acid (polymeric) compounds synthesized in Example 1 having a sugar chain introduction rate of 1%, 5% and 10% were confirmed by NMR and capillary electrophoresis. FIG. 4 shows the results of capillary electrophoresis of a 1% sugar chain-introduced polyglutamic acid compound and polyglutamic acid, and FIG. 5 shows the results of ¹ H-NMR of the 1% sugar chain-introduced polyglutamic acid compound and polyglutamic acid. ^{In 1} H-NMR, each signal intensity ratio between terminal methyl of arachidic acid introduced into Lys and glutamic acid α-H was 0.03H (CH 3): 1H. This supported that the sugar chain introduction rate was 1%. In electrophoresis, no tailing was observed, and a clear difference was observed from PGA as a raw material. The characteristics of 1% sugar chain introduction rate polyglutamic acid are shown below.
-Amount of sugar chain introduced: 5.8 per molecule (= 560Glu × 5%)
Average molecular weight: 116,520 (= 84,600 + (1140 × 28))
Number of moles of sugar chain per μg: 1 [μg] /116,520×5.6 [Glu] = 6.15 × 10 ⁻⁵ [μmol]

Capillary electrophoresis results of 5% sugar chain introduced polyglutamic acid compound (sugar chain introduction rate 5% polyglutamic acid compound) and polyglutamic acid are shown in FIG. 6, and ¹ H-NMR results of 5% sugar chain introduced polyglutamic acid compound and polyglutamic acid are shown in FIG. Is shown in FIG. ^{In 1} H-NMR, each signal intensity ratio between terminal methyl of arachidic acid introduced into Lys and glutamic acid α-H was 0.18H (CH 3): 1H. This supported that the sugar chain introduction rate was about 5%. In electrophoresis, the sugar chain introduction rate increased and tailing was observed. Moreover, a slight difference was seen from PGA which is a raw material. The characteristics of the polyglutamic acid compound with a sugar chain introduction rate of 5% are shown below.
-Amount of sugar chain introduced: 28 per molecule (= 560Glu × 5%)
Average molecular weight: 116,520 (= 84,600 + (1140 × 28))
Number of moles of sugar chain per μg: 1 [μg] / 116,520 × 28 [Glu] = 2.40 × 10 ⁻⁴ [μmol]

FIG. 8 shows the results of capillary electrophoresis of 10% sugar chain-introduced polyglutamic acid compound and polyglutamic acid, and FIG. 9 shows the results of ¹ H-NMR of the 10% sugar chain-introduced polyglutamic acid compound and polyglutamic acid. ^{In 1} H-NMR, each signal intensity ratio of terminal methyl of arachidic acid introduced into Lys and glutamic acid α-H was 0.32H (CH 3): 1H. This supported that the sugar chain introduction rate was 10%. In electrophoresis, the sugar chain introduction rate increased and tailing was observed. Moreover, the clear difference was seen with PGA which is a raw material. The characteristics of a polyglutamic acid compound having a sugar chain introduction rate of 10% are shown below.
-Amount of sugar chain introduced: 56 per molecule (= 560Glu × 10% )
Average molecular weight: 148,840 (= 84,600 + (1140 × 56))
Number of moles of sugar chain per μg: 1 [μg] / 148,840 × 56 [Glu] = 3.77 × 10 ⁻⁴ [μmol]
The weight of only the sugar chain in 1 μg (NeuLacNAc, molecular weight 674) was 0.04 μg, 0.16 μg and 0.25 μg at the introduction rates of 1%, 5% and 10%, respectively.

  Hereinafter, the compound of the present invention which is a polyglutamic acid compound having a sugar chain introduction rate of 1, 5 or 10% is also simply referred to as a polyglutamic acid compound having a sugar chain introduction rate of 1, 5 or 10%.

The properties of the compounds of the present invention were examined by the following method.
[Experimental material]
1) Viruses Viruses are type A 2009 pandemic strain A / Narita / 1/2009 (H1N1), seasonal viruses A Hong Kong type strain A / Yamaguchi / 20/2006 (H1N1) and A Soviet type A / Aichi / 75 / 2008 (H3N2), type B clinical isolate B / Aichi / 3/2008 (64HAU). Lewis lung carcinoma-monkey kidney (LLC-MK2) cultured at 37 ° C. in 5% Fetal bovine serum (FBS, SIGMA) Minimum essential medium (MEM, Invitrogen) medium, 5% CO ₂ After inoculating the cells and infecting for 1 hour at room temperature, 5% CO in MEM medium containing 1 μg / ml acetyltrypsin (SIGMA) and 0.1% Bovine Serum Albumin (BSA, Nacalai Tesque) _The culture was performed at 34 ° C. for 3 days under _two conditions. The culture supernatant containing the virus was centrifuged (25,000 rpm) at 4 ° C. for 2 hours. After removing the supernatant, the precipitated virus is suspended in a small amount of Phosphate buffered saline (PBS), overlaid with a 50% glycerol-PBS solution, centrifuged at 4 ° C. for 2 hours (38,000 rpm), and then suspended in PBS. Used cloudy. Alternatively, the virus culture and purification method was carried out in the same manner as described in JP-A-2006-241024 and other documents.

[Cytotoxicity test]
The cytotoxicity of the compounds of the present invention was examined by the following method.
1) Method MDCK cells were detached with trypsin (trypsin-EDTA, manufactured by Invitrogen), the number of cells was calculated with a hemocytometer, and a cell suspension was prepared to 2 × 10 ⁵ cells / ml with a growth medium. . The cell suspension (100 μl / well) was added to a 96-well plate and cultured overnight at 37 ° C. in a CO ₂ incubator. The next day, compound samples (stock: 20 mg / ml) were diluted with media to a concentration of 0.1-10 mg / ml. The medium in the 96-well plate was removed, and 100 μl / well of the diluted sample solution was added. As a control, a medium to which no sample was added was used. A three-well sample was prepared and used for each compound sample concentration. After 72 hours, the number of viable cells was calculated by the trypan blue exclusion method. When the number of control cells was taken as 100%, the% of the sample addition group was calculated, and the 50% growth inhibitory concentration (CC ₅₀ ) was determined on a semilogarithmic graph.

2) Results No cytotoxicity was observed at a concentration of 0 to 1 mg / ml, and the CC _{50 of the} compound sample was 6.5 ± 0.14 mg / ml.

[Red blood cell aggregation inhibitory activity (HA inhibitory activity) test]
The HA inhibitory activity of the compounds of the present invention against the following influenza strains was examined using hemagglutination inhibition activity as an index; 2009 pandemic strain A / Narita / 1/2009 (H1N1), the same subtype of human infectivity as pandemic Seasonal influenza virus A / Aichi / 28/2008 (H1N1) (A / HongKong type), A / Aichi / 75/2008 (H3N2) subtype of human infectious seasonal virus (A / Soviet type) different from pandemic .

1) Method Viral hemagglutination inhibition activity was performed as follows. Measurement of hemagglutination inhibition activity was performed using a 96-well microplate (company name: BD Falcon (registered trademark); product name: Microtest (registered trademark) 96-well Assay Plate, Black Flat Bottom, Enhanced Surface, Nonsterile No Lid), guinea pig erythrocytes. Used. In addition, fetuin was used as a positive control. Each virus turbid solution (4HAU) was added to the compound of the present invention (polyglutamic acid compound having a sugar chain introduction rate of 5%) or fetuin in a 0.01% (w / v) gelatin-containing PBS solution (initial concentration 2 mM, serially diluted in doubles). (4 erythrocyte agglutination units) was added in an amount of 100 μl, and the mixture was incubated at 4 ° C. for 1 hour, 0.5% guinea pig erythrocyte-PBS turbid solution was added dropwise (0.5 mL / well), and the mixture was further incubated at 4 ° C. for 1 hour. Aggregation inhibitory activity was determined by examining the concentration of the compound that completely inhibits aggregation, that is, the minimum inhibitory concentration of guinea pig hemagglutination.

2) Results Table 2 shows the minimum inhibitory concentration of guinea pig hemagglutination. It shows that inhibition activity is so high that a value is small.

  The compound of the present invention (polyglutamic acid compound having a sugar chain introduction rate of 5%) was used for all human clinical isolates type A influenza virus (type A 2009 pandemic strain (H1N1), seasonal strain (A Hong Kong type H3N2, A Soviet H1N1) hemagglutination, ie, hemagglutinin (HA) function, was inhibited, and in particular, it showed strong hemagglutination inhibitory activity against the 2009 pandemic strain and also against the seasonal influenza virus strain as a positive control. It showed stronger hemagglutination inhibitory activity than fetuin, whereas it did not show hemagglutination inhibitory activity against avian (duck) virus.

[Sialidase inhibitory activity test]
The sialidase inhibitory activity of the compounds of the present invention against the following viruses was investigated; human clinical isolates type A influenza virus (type A 2009 pandemic strain (H1N1) or seasonal strain (A Hong Kong type H3N2, A Soviet type H1N1) .

1) Method 96-well microplate for fluorescence measurement (company name BD Falcon (registered trademark); product name Microtest (registered trademark)) 96-well Assay Plate, Black Flat Bottom, Enhanced Surface, Nonsterile No Lid) 2 μl of virus-PBS suspension diluted to 20 μg / ml with 100 mM acetate buffer (pH 4.6) and 6.67 × 10 ⁻² to 1 μl of a test compound suspension diluted between 6.67 × 10 ⁻⁶ mg / mL and 1 μl of ultrapure (milli-Q water) were mixed and allowed to react on ice for 1 hour. Furthermore, after adding 1 μl of 0.4 mM 4-MU and stirring with a shaker, the mixture was reacted at 37 ° C. for 30 minutes, and 100 μl of 100 mM sodium carbonate buffer (pH 10.8) was added to stop the reaction. Each reaction solution was measured at an excitation wavelength of 355 nm and a fluorescence wavelength of 460 nm using a multi-label counter (company name: Perkin Elmer Life Science Japan Co., Ltd .; device name: Wallac 1420 ARVOsx multi-label counter). As the test compound, the compound of the present invention containing 1% sugar chain introduced polyglutamic acid, the compound of the present invention containing 5% sugar chain introduced polyglutamic acid, and the compound of the present invention containing 10% sugar chain introduced polyglutamic acid were used.

2) Results The results are shown in FIG. All of the test compounds strongly inhibited sialidase inhibitory activity against all influenza viruses of type A 2009 pandemic strain (H1N1), A Hong Kong type H3N2, and A Soviet type H1N1. The sialidase inhibitory activity against each virus of the compound of the present invention (polyglutamic acid compound having a sugar chain introduction rate of 5%) at a concentration of 0.07 mg / mL (70 ug / ml, molecular weight 100,000: 0.7 nM) in FIG. Indicates. Further, the graph of FIG. 11B shows the relationship between the test compound concentration and the inhibitory activity in the seasonal virus A Soviet type (A / Aichi / 75/2008 (H3N2)). IC ₅₀ for seasonal virus A Soviet type (A / Aichi / 75/2008 (H3N2)) is polyglutamic acid compound with a sugar chain introduction rate of 1%, 0.0080 mg / ml, and polyglutamic acid compound with a sugar chain introduction rate of 5%. The polyglutamic acid compound having a sugar chain introduction rate of 10% was 0.0022 mg / ml and 0.0022 mg / ml. It was found that the compound of the present invention effectively exhibits sialidase inhibitory activity at low concentrations.

The IC ₅₀ of the compound of the present invention (polyglutamic acid compound having a sugar chain introduction rate of 5%) with respect to the 2009 pandemic strain was ˜60 μg / ml, ie, on the order of 0.6 nM. Also, the IC ₅₀ for seasonal strains (A Hong Kong type, H3N2; A USSR type, H1N1) was 2.2-8.0 μg / ml, that is, 0.022-0.08 nM order. These can be said to have high sialidase inhibitory activity 10 to 100 times that of Tamiflu and Relenza, both of which have an IC ₅₀ of the order of 1 nM.

[Binding test with influenza]
Binding of the compounds of the present invention to each of the following influenza viruses was investigated: 2009 pandemic strain (A / Narita / 1/2009 (H1N1)), seasonal influenza virus strain (A / Yamaguchi / 20/2006 (H1N1)) Seasonal influenza virus strain (A / Aichi / 75/2008 (H3N2)), type B clinical isolate (B / Aichi / 3/2008).

1) Method To each well of a microtiter plate, an ethanol-diluted solution (50 μl) of a polyglutamic acid compound with a sugar chain introduction rate of 5% at 0 to 200 ng / well is added, and the plate is allowed to stand at 37 ° C. to evaporate ethanol. I let you. Next, 200 μl of a phosphate buffer (pH 7.0) containing 5% bovine serum albumin was added to each well and allowed to stand for 12 hours or more. After washing this plate with phosphate buffer, 50 μl of each human influenza virus suspension prepared to 64 HAU with phosphate buffer containing 0.2% BSA was added to each well for 12 hours at 4 ° C. Reacted. After washing the wells with phosphate buffer, the sialidase activity of the virus bound to the plate was measured by the same method as described above.

2) Results The results are shown in FIGS. As a result, the compound of the present invention (polyglutamic acid compound having a sugar chain introduction rate of 5%) was found to be a 2009 pandemic strain (A / Narita / 1/2009 (H1N1)), seasonal influenza virus strain (A / Yamaguchi / 20 / It was shown to bind to 2006 (H1N1), seasonal influenza virus strain (A / Aichi / 75/2008 (H3N2)), and B-type clinical isolate (B / Aichi / 3/2008).

  From this result, it was found that the compound of the present invention not only suppresses the growth of influenza virus in the living body but also can adsorb before the virus enters the living body.

[Infection inhibitory concentration test]
Infectious inhibitory concentrations of the compounds of the present invention against the following influenza viruses were investigated: 2009 pandemic strain A / Narita / 1/2009 (H1N1), seasonal strain A / Yamaguchi / 20/2006 (H1N1), seasonal strain A / Aichi / 75/2008 (H3N2), laboratory strain B / Lee / 40, clinical isolate B / Aichi / 3/2008 (Yamagata like strain), avian low pathogenic strain A / Mallard / Hokkaido / 24 / 2009 (H5N1), seasonal strain A / Aichi / 102/2008 (H3N2).

Viral infection inhibition assay was performed as follows using the method of Nongluk Sriwilaijaroen, et al., Food Chem., 127 (2011) 1-9).
1) Material (MDCK SIAT-1 cells)
A sialic acid transferase SIAT-1 gene (drug antibiotic 418 is added and cultured) is introduced into MDCK cells, and the human receptor sugar chain Neu5Acα2- of influenza virus is introduced. It is a cell in which a 6Gal-linked sugar chain is forcibly expressed.

1) Method
MDCK SIAT-1 cells were cultured until confluent (this was called confluent cells). The culture solution was removed from the confluent cells, and the serum and antibiotic G418 contained in the culture solution were removed. Confluent cells and viruses (eg, A / Narita / 1/2009 H1N1, MOI: 0.03 Plaque-forming units (pfu) / cell) with different amounts of inhibitors, 2 mM L-glutamine and penicillin (100 U / ml) -streptomycin ( Incubated with MEM medium containing 100 mg / ml for 1 hour at 37 ° C. or 4 ° C., respectively. After 1 hour, the inhibitor-containing medium is removed from the confluent cells, replaced with a virus inhibitor medium previously treated with a culture medium containing the inhibitor at 4 ° C. for 1 hour, and incubated in a CO ₂ incubator at 37 ° C. for 1 hour. . Thereafter, the cells are washed once with a medium, a medium supplemented with the same inhibitor concentration and 2 μg / ml acetyltrypsin is added, and the cells are cultured in a CO ₂ incubator for 24 hours at 37 ° C. Thereafter, the infected cells were fixed with methanol, and the viral antigens of the infected cells were fluorescently developed by a fluorometric method (Nongluk Sriwilaijaroen et al., Virology Journal, 6, 124 (2009)) and measured with a DEPDA-CN fluorometer. That is, the inhibitor-treated infected cells and untreated control-infected cells were reacted with the primary antibody mouse anti-influenza virus nucleoprotein antibody (4E6), then reacted with the secondary antibody β-galactosidase labeled anti-mouse antibody and bound. The amount of secondary antibody is measured by adding the substrate 4-methylumbelliferyl-β-galactoside (MU-Gal) and measuring the released 4-methylumbelliferone (MU) at an excitation wavelength of 336 nm and a transmission wavelength of 490 nm. It was measured by.

Further, the virus infection site (infectious foci: viral antigen expression site in infected cells) was determined by using mouse anti-influenza virus nucleoprotein antibody (primary antibody) and HRP (Horseradish peroxidase) -conjugated goat anti-mouse IgG polyclonal antibody (secondary antibody, Nichirei Bioscience) And the bound secondary antibody was dissolved in H ₂ O ₂ and N, N′-diethyl-p-phenylenediamine dihydrochloride (DEPDA) and 4-dissolved in 100 mM citrate buffer (pH 6.0). The color was visualized in blue insoluble in water with chloro-1-naphthol (4-CN), and was visually or photographed.

2) Results Table 3 shows the results.

The compound of the present invention (polyglutamic acid compound having a sugar chain introduction rate of 5%) was tested for all tested influenza viruses (type A: 2009 pandemic virus (H1N1), seasonal virus (H1N1, H3N2), type B virus). Infection was inhibited, and the infection inhibitory concentration (IC ₅₀ ) was 10 ⁻⁷ M (seasonal type A, type B virus) to 10 ⁻⁸ M (2009 A pandemic strain). Furthermore, as described above, the compound of the present invention strongly inhibits the hemagglutinin (HA) spike function of the virus, and also strongly inhibits the sialidase activity of the neuraminidase (NA) spike (on the order of 0.6 nM to 0.02 nM, Tamiflu, About 10 to 100 times stronger than Relenza). That is, the compound of the present invention is a multi-functional inhibitor that inhibits virus infection, adsorption, and budding. Since conventional inhibitors inhibit only neuraminidase spikes, or inhibit only M2 ion channels (not effective for type B), resistant strains are prevalent worldwide. However, since functional molecules associated with HA, NA, and proliferative properties rarely occur at the same time, it can be said that the compound of the present invention has a design in which resistant strains are hardly formed (FIG. 14).

  The present invention can be suitably used in the field of influenza therapeutics and preventives, the field of influenza infection, adsorption, and budding inhibitors, and the field of providing materials for removing influenza viruses from the environment.

Claims (13)

Formula (I) below
(In the formula, S is Neu5Acα2-6Galβ1-4 (3) GlcNAcβ1 structure or Neu5Acα2-3Galβ1-4 (3) GlucNAcβ1 structure-containing hemiglutinin-binding hemiglutinin binding moiety, L is lysine, ornithine , 2,6-diaminohexanoic acid, 3,5-diaminobenzoic acid, 3,4-diaminobenzoic acid, and 1,2-diaminohexanecarboxylic acid. A hydrophobic part which is an alkyl group having 3 to 35 carbon atoms which may have a bond, and P has a chain length of 100 angstroms or more, and is composed of a polyglutamic acid, a polyaspartic acid, and a styrene-maleic acid copolymer. a compound or a salt thereof represented by the representative) a polymer moiety selected, S, C, P it The compound or a salt thereof, wherein the coupling moiety is any of the following 1) to 3) of the record and L.
1) The connecting partial structure of S and L is —NHCO— or —OCO—, the connecting partial structure of C and L is —CONH— or —CH═NH—, and the connecting partial structure of P and L is , -CONH- or -CH = NH-.
2) The connecting partial structure of S and L is —CONH— or —CH═NH—, the connecting partial structure of C and L is —NHCO— or —OCO—, and the connecting partial structure of P and L is , -CONH- or -CH = NH-.
3) The connecting partial structure of S and L is —CONH— or —CH═NH—, the connecting partial structure of C and L is —CONH— or —CH═NH—, and the connecting portion of P and L The structure is —NHCO— or —OCO—. The compound or a salt thereof according to claim 1, wherein the receptor pseudo-sialoglycosaccharide moiety having hemagglutinin binding ability comprises a Neu5Acα2-6Galβ1-4 (3) GlcNAcβ1 structure. The compound or a salt thereof according to claim 1 or 2, wherein the polymer moiety is polyglutamic acid. The linker or the salt thereof according to any one of claims 1 to 3, wherein the linker moiety is lysine. Formula (II) below
Wherein PGA is the formula (III)
Where m and n are integers and (m + 1) n is 50 to 1000. The compound or its salt in any one of Claims 1-4 which is a compound or its salt shown by this. An influenza virus hemagglutinin function and sialidase inhibitor comprising the compound according to any one of claims 1 to 5 or a salt thereof as an active ingredient. 7. Inhibitor according to claim 6, characterized in that the sialidase is neuraminidase. Influenza virus infection and budding, and an adsorption inhibitor to the cell surface, comprising the compound according to any one of claims 1 to 5 or a salt thereof as an active ingredient. The influenza preventive or therapeutic agent which uses the compound or its salt in any one of Claims 1-5 as an active ingredient. An adsorbent for influenza virus comprising the compound according to any one of claims 1 to 5 or a salt thereof as an active ingredient. An adsorbent for influenza virus carrying the compound according to any one of claims 1 to 5 or a salt thereof. A kit for examining a mutation of an influenza virus carrying the compound according to any one of claims 1 to 5 or a salt thereof. A kit for examining the in vivo kinetics of influenza virus comprising the compound according to any one of claims 1 to 5 or a salt thereof as an active ingredient.